Methanogenic patterns in the gut microbiome are associated with survival in a population of feral horses

Gut microbiomes are widely hypothesised to influence host fitness and have been experimentally shown to affect host health and phenotypes under laboratory conditions. However, the extent to which they do so in free-living animal populations and the proximate mechanisms involved remain open questions. In this study, using long-term, individual-based life history and shallow shotgun metagenomic sequencing data (2394 fecal samples from 794 individuals collected between 2013–2019), we quantify relationships between gut microbiome variation and survival in a feral population of horses under natural food limitation (Sable Island, Canada), and test metagenome-derived predictions using short-chain fatty acid data. We report detailed evidence that variation in the gut microbiome is associated with a host fitness proxy in nature and outline hypotheses of pathogenesis and methanogenesis as key causal mechanisms which may underlie such patterns in feral horses, and perhaps, wild herbivores more generally.


Supplementary Note 1
Sable Island Horse Population Ecology.Horses were introduced to Sable Island in the mid-1700s 1 .In the intervening centuries Sable Island horses have been left free-living; although, additional intermittent introduction of horses from the mainland occurred as recently as 1940.
Horses were similarly infrequently taken from Sable Island to be sold on the mainland until 1960, whereupon the Sable Island feral horse population was granted legal protections under the Sable Island Regulations of the Canada Shipping Act.Based on aerial surveys conducted over the decade after receiving protected status, the Sable Island horse population varied from 133-232 horses between 1961-1970 2  Horses occupy the entire vegetated length and width of the island.This population lacks predators, and so, is primarily bottom-up regulated by access to food and freshwater 2,3 .Nearly all mortality in this population occurs during the winter and early-spring, when horse energy reserves are low, and quality forage is unavailable.Marram grass is among the most abundant and widespread plant species on Sable Island and comprises a large portion of the horse diet; however, the semi-succulent plant sea sandwort is preferred where locally available at the eastern and western extremities of the island (pers.comm.K. Johnsen).Where permanent freshwater ponds are absent towards the eastern and western ends of Sable Island, horses are obliged to dig wells.
The availability of freshwater ponds towards the longitudinal midpoint of the island influence horse movement, catalyze social interactions, and shape the social structure of the population, which segregates into 65-144 mixed-sex social bands or groups of bachelor males 4,5 .
Mixed-sex social bands and bachelor male groups occupy spatially overlapping homeranges.However, the Sable Island horse population is not freely inter-mix, but rather, segregates into two sub-populations in the summer at approximately the longitudinal midpoint, which are occasionally connected by migrants 6 .In the east, horse home-ranges are generally narrower and strongly influenced by the distribution of horse dug wells.In the west, horse home-ranges generally span the western third of the island and are shaped by the bi-directional movement of horses from sea sandwort lawns in the west, to freshwater ponds towards the island interior.
Sable Island horses have a polygynous mating structure wherein females segregate into mixed-sex social bands.Each harem of females is guarded by a dominant male (stallion), who monopolizes breeding opportunities by preventing mating incursions by bachelor males or competing band stallions.Males unable to secure a harem (a group of reproductively mature females) are solitary or form less stable social groups with other bachelor males.More rarely, subordinate males are tolerated by band stallions if they participate in harem defense 7 .Between 50-80 mixed-sex social bands are observed on Sable Island annually, and across years, the average harem size is 2-3 females of reproductive age 4 .
The population density of horses decreases from west to east on Sable Island 8 , as does average horse body condition, intestinal parasitic nematode burden (estimated from fecal egg counts) 9 , and the tolerance of horses to human presence 10 .Bacterial richness in the horse fecal microbiome similarly declines from west to east, however, horse access to low-fibre sea sandwort at the eastern and western tips of the island is also associated with a decrease in richness, as well as differences in fecal microbiome structure 11 .

Supplementary Note 2
Sable Island National Park Reserve Description.Sable Island is a narrow oceanic sandbar, 160 km from the nearest landfall in eastern Nova Scotia, Canada 12 .The island is 1.5 km wide near the midpoint but stretches 42 km from east to west in near perfect perpendicularity to global meridians.
Positions of longitude therefore provide an informative 1-dimensional proxy for spatial relationships on the island.Between 1801-1958, Sable Island was populated by a small community that operated a provincial life-saving station, which prevented shipwrecks, or rescued shipwreck survivors.Since 1958, Sable Island has lacked permanent human settlement, but has been intermittently peopled by Government of Canada staff and natural sciences researchers.
Three major environmental gradients dominate Sable Island: (1) The Atlantic Ocean abuts contiguous sandy beaches on the north and south shores of the island along its entire length.Moving from shorelines towards the latitudinal midpoint of the island, barren beaches give way to American sea-rocket (Cakile edentula) dotted mounds that lay at the base of more densely marram grass (Ammophila breviligulata) and beach pea (Lathyrus japonicus) vegetated barrier dunes (10-30 m in height) 12 .Barrier dunes to the north and south shelter late-succession heathland community valleys in the interior of the island 13 .
(2) Sheltered heathland valleys and freshwater ponds are more common towards the longitudinal midpoint of the island, where the barrier dunes are tallest 14 .As the island tapers towards its longitudinal extremes, heathland valleys and barrier dunes give way to lower-lying marram and beach pea plateaus.Dense lawns of sea sandwort (Honkenya peploides) bookend the eastern and western tips of the island and are the only locations where this plant species is observed in abundance 14 ; although, sea sandwort lawns were locally extirpated from the eastern side of the island by 2017, when a nearly 5 km segment of Sable Island became submerged by the Atlantic Ocean 12 .
(3) Average dune height increases from west to east and vegetation density decreases along the same axis 15 .

Supplementary Fig. 2 .
Patterns of repeatability in gene family abundance in the Sable Island horse fecal microbiome.Within-individual repeatability of centred log ratio (CLR) transformed gene hit abundance in relation to the estimated association between a one standard deviation increase in the CLR-transformed abundance of a given gene family and the odds of horse survival.Each gene family is represented by a single point.Solid red line denotes the best fit 2 nd order polynomial from a two-sided general linear model (1416 gene families from 2394 samples spanning 794 individuals) with 95% confidence interval shading (linear term: β = 0.30 ± 0.04, t = 6.736, p < 0.001; quadratic term: β = 0.27 ± 0.04, t = 6.109, p < 0.001).Source data are provided as a Source Data file.Supplementary Fig. 9. Associations of microbiota with the odds of horse survival from binomial generalized linear mixed models in the full dataset (2394 samples from 794 individuals) and among a subset of samples collected from east of −60.08º (2069 samples from 747 individuals).Dashed diagonal denotes the 1:1.Microbiota (read pairs classified to the finest resolution possible, strain to phylum) are represented by points coloured by major taxonomic grouping.Source data are provided as a Source Data file.